MECHANISMS OF INDUCTION OF SKIN CANCERS BY UV LIGHT

描述：这项修订后的申请寻求四年的研究支持 紫外线辐射诱发小鼠皮肤癌的分子机制。这个 细胞和分子过程发生在最初的 致癌物-细胞相互作用和肿瘤生长的开始在很大程度上 未知。以前的研究已经证明了p53肿瘤的突变 抑制基因，并在一定程度上存在于人和人的ras癌基因中 紫外线诱导的小鼠皮肤癌。然而，目前还不知道这些是否 突变发生在紫外线皮肤癌的早期或晚期。目标 这项提议的目的是在细胞和细胞上识别时间变化 分子水平，发生在紫外线皮肤癌的过程中，并确定 这些改变是否导致非调控的凋亡细胞死亡 表皮。假设是P53肿瘤抑制基因的改变 基因是早期事件，凋亡细胞死亡和 细胞凋亡调控基因参与肿瘤的发生和发展 紫外线诱发的皮肤癌。具体目标是：(1)确定是否 紫外线诱导的ras和p53突变是皮肤发生的早期或晚期事件。 肿瘤的发生；(2)确定细胞凋亡的失调 有助于紫外线诱发皮肤癌的发生；(3)确定 紫外线诱导的小鼠皮肤肿瘤是否存在特异性基因改变 细胞影响其对干扰素或肿瘤坏死的敏感性 因子诱导的细胞凋亡。为了达到这些目的，在目标1中，紫外线照射的小鼠皮肤 将在皮肤癌发生过程中的进行性时间点进行分析 等位基因特异性聚合酶链式反应检测RAS和P53基因突变 通过单链构象多态性分析和核苷酸 测序。在目标2中，诱导晒伤(凋亡)细胞在 紫外线照射小鼠皮肤癌变过程中将进行原位测量 通过原位末端标记法检测细胞凋亡调控基因的表达 P53、Bcl2和Bax将通过免疫印迹法检测 抗体。在目标3中，特定的基因改变之间的关系 紫外线诱导的小鼠皮肤癌及其易感性 干扰素-γ或肿瘤坏死因子诱导的细胞凋亡 通过四甲基偶氮唑盐染料摄取、DNA片段化和染色质研究 冷凝分析。

DESCRIPTION: This revised application seeks 4 years' support for studies of molecular mechanisms by which UV radiation induces skin cancer in mice. The cellular and molecular processes that occur during the initial carcinogen-cell interaction and the onset of tumor growth are largely unknown. Previous studies have demonstrated mutations in the p53 tumor suppressor gene, and to a certain extent, in ras oncogenes in both human and UV-induced mouse skin cancers. It is, however, not known whether these mutations arise early or late during UV skin carcinogenesis. The objectives of the proposal are to identify the temporal changes, at the cellular and molecular level, that occur during UV skin carcinogenesis, and to determine whether these alterations result in deregulated apoptotic cell death in the epidermis. The hypothesis is that alterations in the p53 tumor suppressor gene are early events and that dysregulation of apoptotic cell death and apoptosis regulatory genes contribute to the development and progression of UV-induced skin cancer. The specific aims are: (1) To determine whether the UV-induced ras and p53 mutations are early or late events during skin carcinogenesis; (2) to determine whether dysregulation of apoptosis contributes to the development of UV-induced skin cancer; (3) to determine whether specific genetic alterations present in UV-induced mouse skin tumor cells influence their susceptibility to interferon-gamma- or tumor necrosis factor-induced apoptosis. To these ends, in Aim 1, UV-irradiated mouse skin will be analyzed at progressive time points during skin carcinogenesis for ras and p53 mutations by allele-specific polymerase chain reaction followed by single-strand conformation polymorphism analysis and nucleotide sequencing. In Aim 2 the induction of sunburn (apoptotic) cells in UV-irradiated mouse skin during skin carcinogenesis will be measured in situ by the TUNEL method, and expression of apoptosis regulatory genes such as p53, Bcl-2, and Bax, will be determined by western blotting using specific antibodies. In Aim 3 the relationship between specific genetic alterations present in UV-induced mouse skin cancers and susceptibility to interferon-gamma- or tumor necrosis factor-induced apoptosis will be investigated by MTT dye uptake, DNA fragmentation, and chromatin condensation assays.